Rubredoxin from the hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10 ؊6 s ؊1 at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the Ϸmillisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 10 4 and 10 6 , for which EX2 exchange kinetics were directly verified, have exchange activation energy values within 2-3 kcal͞mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.
Hydroxide-catalyzed exchange rate constants were determined for those amides of FK506-binding protein (FKBP12), ubiquitin, and chymotrypsin inhibitor 2 (CI2) that are solvent-accessible in the high-resolution X-ray structures. When combined with previous hydrogen exchange results for the rubredoxin from Pyrococcus furiosus, the acidity of these amides was calculated by continuum dielectric methods as a function of the nonpolarizable electrostatic parameter set, internal dielectric, and the charge distribution of the peptide anion. The CHARMM22 parameter set with an internal dielectric value of 3 and an ab initio-derived anion charge distribution yielded an rmsd value of 7 for the 56 amide exchange rate constants ranging from 10(0.67) to 10(9.0) M(-1) s(-1). The OPLS-AA parameter set yielded comparably robust predictions, while that of PARSE, AMBER parm99, and AMBER ff03 performed more poorly. The small value for the optimal internal dielectric, combined with the brief lifetime of the peptide anion intermediate and the uniformity of the correlation between predicted and observed amide acidities, is consistent with electronic polarizability providing the dominant contribution to dielectric shielding. By construction, nonpolarizable force fields do not model electric field attenuation by electronic polarizability. Accurate prediction of the total electrostatic energy by such force fields necessitates the hyperpolarization of the atomic charge values in order to match the average electric field energy density (1/2)epsilon(tau)E(2)(tau) when epsilon(tau) is set to the in vacuo dielectric value of 1. The resulting predictions of the experimental hydrogen exchange data demonstrate the substantial systematic errors in the predicted electrostatic potential that can arise when dielectric shielding due to electronic polarizability is neglected.
The exchange rates of the static solvent-accessible amide hydrogens of Pyrococcus furiosus rubredoxin range from near the diffusion-limited rate to a billion-fold slower for the non-hydrogen-bonded Val 38 (eubacterial numbering). Hydrogen exchange directly monitors the kinetic acidity of the peptide nitrogen. Electrostatic solvation free energies were calculated by Poisson-Boltzmann methods for the individual peptide anions that form during the hydroxide-catalyzed exchange reaction to examine how well the predicted thermodynamic acidities match the experimentally determined kinetic acidities. With the exception of the Ile 12 amide, the differential exchange rate constant for each solvent-exposed amide proton that is not hydrogen bonded to a backbone carbonyl can be predicted within a factor of 6 (10 (0.78)) root-mean-square deviation (rmsd) using the CHARMM22 electrostatic parameter set and an internal dielectric value of 3. Under equivalent conditions, the PARSE parameter set yields a larger rmsd value of 1.28 pH units, while the AMBER parm99 parameter set resulted in a considerably poorer correlation. Either increasing the internal dielectric value to 4 or reducing it to a value of 2 significantly degrades the quality of the prediction. Assigning the excess charge of the peptide anion equally between the peptide nitrogen and the carbonyl oxygen also reduces the correlation to the experimental data. These continuum electrostatic calculations were further analyzed to characterize the specific structural elements that appear to be responsible for the wide range of peptide acidities observed for these solvent-exposed amides. The striking heterogeneity in the potential at sites along the protein-solvent interface should prove germane to the ongoing challenge of quantifying the contribution that electrostatic interactions make to the catalytic acceleration achieved by enzymes.
The substrate-bound form of the enzyme heme oxygenase (HO), which catalyzed the stereospecific alpha-meso bridge cleavage of hemin to yield biliverdin IX alpha, has been investigated by 1H NMR in both its primarily high-spin and its cyanide-inhibited low-spin forms. Both derivatives yield 1H NMR spectra indicative of extensive heterogeneity that is largely resolved when a 2-fold-symmetric hemin substrate is bound. The structural origin of the heterogeneity is shown to result from approximately 1:1 isomeric binding of the native hemin substrate in the binding pocket. The substrate orientational disorder is about the alpha,gamma-meso axis, as established on the basis of 2D NMR experiments that identify characteristic aromatic van der Waals contact in the substrate binding pocket. The isomeric substrate-HO complexes exhibit differential cyanide affinity, and the ratio of isomers is sensitive to the hemin 2,4-substituents. The assignment of hemin signals by isotopic labeling and 2D NMR methods reveals a contact shift pattern that reflects an unusual hemin electronic structure that is characterized by large differences in delocalized spin density for the two positions within a given pyrrole, rather than the more conventional large differences between adjacent pyrroles. This pattern of spin density delocalized primarily to the pyrrole positions adjacent to the alpha,gamma-meso axis can be rationalized by postulating a direct electronic perturbation of the hemin by the protein matrix in the form of an anionic side chain close to the alpha-meso carbon. Similar influences on hemin electronic structure, in the form of chemical substitution of the meso positions, have been observed in iron porphyrin compounds and successfully modeled by simple molecular orbital theory (Tan et al., 1994). This is interpreted as evidence for a direct electronic effect by HO to activate the alpha-meso position for electrophilic rather than nucleophilic attack. The unique contact shift pattern is present to different degrees for the two hemin orientations, is strongly pH dependent, and is largely abolished at acidic pH. Portions of several heme pocket residues are located and it is shown that the pattern of the dipolar shifts for these residues, which likely reflects the distal steric influence on the tilt of the coordinated cyanide, differs significantly for the two substrate orientations.(ABSTRACT TRUNCATED AT 400 WORDS)
The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 (FK506-binding protein of 12 kDa) contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43°C. The largest differences in chemical shift occur for the 80′s loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the β-sheet, across the active site to the α-helix and into the 50′s loop. Each of the seven proline residues adopts a trans-peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational line-broadening transition(s) that occur ~105-fold more rapidly, proposed previously to arise from a single global process. The 1.70 Å (1 Å=0.1 nm) resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the line-broadening transition for residues near the 80′s loop. Line-broadening was also decreased for the residues in the α-helix and 50′s loop, whereas line-broadening in the 40′s loop was unaffected. The K44V mutation selectively reduces the line-broadening in the 40′s loop, verifying that at least three distinct conformational transitions underlie the line-broadening processes of FKBP12.
Paramagnetically induced relaxation effects of O2 and the nitroxide 4-hydroxy TEMPO were measured for the amide protons of perdeuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium pasteurianum. For both O2 and the impermeant nitroxide, the induced relaxation at the static solvent inaccessible amide sites is dominated by long-range interactions with the paramagnetic species in the bulk aqueous phase. The upper bound of O2 solubility in the internal matrix of the rubredoxins is one-tenth that of the bulk aqueous phase. Furthermore, the difference between the oxygen solubilities inside the two rubredoxins is at most 1% that of bulk water O2 solubility, suggesting that there are only modest differences in this measure of fluidity for the mesophile vs hyperthermophile protein interiors. Calculations based on the assumption of a paramagnet uniformly distributed on the protein exterior yield accurate predictions at nearly all amide sites for the minimum relaxation value observed from either the O2 or nitroxide data. Model calculations indicate that the readily obtained paramagnetically induced relaxation effects should prove effective in recognition of structural homology for proteins that are too widely diverged for sequence-based recognition.
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